Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines

Cheok Wui Chee; Nor Hisam Zamakshshari; Vannajan Sanghiran Lee; Iskandar Abdullah; Rozana Othman; Yean Kee Lee; Najihah Mohd Hashim; Nurshamimi Nor Rashid

doi:10.1371/journal.pone.0270970

Peer Review History

Original SubmissionOctober 17, 2021
14 Feb 2022 Decision Letter - Chakrabhavi Dhananjaya Mohan, Editor PONE-D-21-32928Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines.PLOS ONE Dear Dr. Nor Rashid, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 31 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Search for new phytochemicals, with anti-cancer properties is an ever green area of research in the field of drug discovery, In view of this the authors in the present manuscript entitled “Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines” have studied the Anti-cancer potentials of root barks of Morinda citrifolia and isolated the eight anthraquinone derivatives. Even though the anticancer properties of anthraquinone compounds from Morinda citrifolia is previously studied in this present work, the authors have identified the key targets of these active compounds by protein docking studies. They have identified the binding sites for these compounds in the three important genes including P53, KRAS and β- catenin which were routinely mutated in colorecral cancer (CRC). Comment #1. In the Cytotoxic assay carried out the authors have used three human colorectal cancer cell lines: HT29 (with p53 and APC mutation), LS174T (with KRAS mutation) and HCT116 (with KRAS mutation). In this study HCT116 wells were seeded at 7500 cells/well while other cells were seeded at 30,000 cells/well. What is the reason for varying the number of cells seeded in the other cell lines? Comment #2. In the Introduction part in the last paragraph one of the reference needs to be corrected. It is written as (et al., 2016), without the author’s name. mention the author’s name as well. Comment #3. In the figures 3,4,5,6 representing the Cytotoxicity effect of anthraquinone compounds 1-8 in different cancer cell lines and normal cells, the graph could be represented in a colored line, which makes the lines distinguishable clearly. The authors have identified that Morindone has high cytotoxicity effect and great selectivity index towards colorectal cancer cell lines as well as strong binding affinities in in-vitro and in-silico investigation towards multiple protein targets of β-catenin, MDM2-p53, and KRAS in comparison to other anthraquinones. In detail Molecular docking studies have shown that all the active compounds can bind well into each targeted protein receptor by binding to important residues in the protein, explaining their inhibitory activities. Further in vivo assays with Morindone are needed for the development of potential lead compound against colorectal cancer cells. Overall, the manuscript could be accepted for publication once the above comments are considered and thoroughly revised again for any topographical error. Reviewer #2: Review comments Manuscript Number: PONE-D-21-32928 Dr. Nurshamimi and coworkers describes the Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines. The work carried out by this group is extension work of Kamiya group but this group used three colorectal cancer cell lines and activity shown by all the three cell lines was good. After isolation and in vitro studies, binding efficiency of all the isolated derivatives were investigated. From the studies it is confirmed that Morindone is showed moderate inhibitory activity against multitargets like β-catenin, MDM2-p53 and KRAS. 1) Provided IR stretching frequencies isolated anthraquinone derivatives is not clear. All the derivatives with hydroxyl group but authors not mentioned the stretching frequencies of in data. 2) Include 1H NMR and 13C NMR data of active molecule Morindone in the manuscript. 3) Include in vivo assay results. After all these modification paper will be considered. I am suggesting major revision. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Plos one review comments.docx https://doi.org/10.1371/journal.pone.0270970.r001
Revision 1
24 Mar 2022 Author Response Response to reviewers Title: Morindone from morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines Reference number: PONE-D-21-32928 Reviewer #1: Search for new phytochemicals, with anti-cancer properties is an ever green area of research in the field of drug discovery, In view of this the authors in the present manuscript entitled “Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines” have studied the Anti-cancer potentials of root barks of Morinda citrifolia and isolated the eight anthraquinone derivatives. Even though the anticancer properties of anthraquinone compounds from Morinda citrifolia is previously studied in this present work, the authors have identified the key targets of these active compounds by protein docking studies. They have identified the binding sites for these compounds in the three important genes including P53, KRAS and β- catenin which were routinely mutated in colorecral cancer (CRC). Comment #1. In the Cytotoxic assay carried out the authors have used three human colorectal cancer cell lines: HT29 (with p53 and APC mutation), LS174T (with KRAS mutation) and HCT116 (with KRAS mutation). In this study HCT116 wells were seeded at 7500 cells/well while other cells were seeded at 30,000 cells/well. What is the reason for varying the number of cells seeded in the other cell lines? Response: The cell seeding density for each cell line has been optimized according to their proliferation rate and confluency at the end of treatment, hence the differences. HCT 116 cells showed faster proliferation rate while the other cells showed slower proliferation rate. Comment #2. In the Introduction part in the last paragraph one of the reference needs to be corrected. It is written as (et al., 2016), without the author’s name. mention the author’s name as well. Response: Thank you for the comment. This has been updated with Vancouver style referencing, which is the PloS One reference style. The changes can be tracked in the Introduction section in the revised manuscript. Comment #3. In the figures 3,4,5,6 representing the Cytotoxicity effect of anthraquinone compounds 1-8 in different cancer cell lines and normal cells, the graph could be represented in a colored line, which makes the lines distinguishable clearly. Response: Thank you for the suggestion. We have changed them to the colored line graph in Figures 4 – 7. The authors have identified that Morindone has high cytotoxicity effect and great selectivity index towards colorectal cancer cell lines as well as strong binding affinities in in-vitro and in-silico investigation towards multiple protein targets of β-catenin, MDM2-p53, and KRAS in comparison to other anthraquinones. In detail Molecular docking studies have shown that all the active compounds can bind well into each targeted protein receptor by binding to important residues in the protein, explaining their inhibitory activities. Further in vivo assays with Morindone are needed for the development of potential lead compound against colorectal cancer cells. Overall, the manuscript could be accepted for publication once the above comments are considered and thoroughly revised again for any topographical error. Reviewer #2: Review comments Manuscript Number: PONE-D-21-32928 Dr. Nurshamimi and coworkers describes the Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines. The work carried out by this group is extension work of Kamiya group but this group used three colorectal cancer cell lines and activity shown by all the three cell lines was good. After isolation and in vitro studies, binding efficiency of all the isolated derivatives were investigated. From the studies it is confirmed that Morindone is showed moderate inhibitory activity against multitargets like β-catenin, MDM2-p53 and KRAS. 1) Provided IR stretching frequencies isolated anthraquinone derivatives is not clear. All the derivatives with hydroxyl group but authors not mentioned the stretching frequencies of in data. Response: The IR stretching frequencies data of all 8 anthraquinone compounds has been published in Zamakshshari et al., 2017 – Cytotoxic activities of anthraquinones from morinda citrifolia towards SNU-1 and LS-174-T and K562 cell lines. Since this work is a continuation from Zamakshshari et al., 2017, we would think that there is no need to provide it again. 2) Include 1H NMR and 13C NMR data of active molecule Morindone in the manuscript. Response: The data is provided in the supporting information file named S9_Table.pdf. This change can be tracked in the Supporting Information section in the revised manuscript. 3) Include in vivo assay results. Response: We are looking forward to test this morindone using animal model. However, due to the limitation of the current funding, we are not able to test the morindone using animal model. Therefore, the in vivo work will be our next step, once we receive the upcoming funding for the study. After all these modification paper will be considered. I am suggesting major revision. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0270970.r002
2 Jun 2022 Decision Letter - Chakrabhavi Dhananjaya Mohan, Editor PONE-D-21-32928R1Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines.PLOS ONE Dear Dr. Nor Rashid, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 17 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Chakrabhavi Dhananjaya Mohan, Ph.D Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have considered the previously suggested corrections and included them in the revised manuscript. The manuscript can now be accepted without need of further revision Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Plos one revision.docx https://doi.org/10.1371/journal.pone.0270970.r003
Revision 2
15 Jun 2022 Author Response Response to reviewers Title: Morindone from morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines Reference number: PONE-D-21-32928 Reviewer #2: In this study, the authors extracted the potential anthraquinone compounds from the root bark of Morinda citrifolia. Eight potential anthraquinone compounds were successfully isolated, purified and tested for their anticancer efficacy. Despite the fact that this work is a repetition of previously published work, authors must provide characterization data as well as scanned images of the active molecule. Comment #1. I have suggested that, provided IR stretching frequencies some of the functional groups are missing, provide an active molecule scanned IR spectra. Response: Thank you very much on your suggestion. We have now provided the active molecule (morindone) IR spectra under figure 8A of the results section. Comment #2. The authors provided data in the table, but I suggest you to include scanned images of H1 NMR and C13 NMR of active molecule. Response: Thank you for the comment. We have now included the scanned images of H1 NMR and C13 NMR of the active molecule (morindone) under figure 8C and 8D, respectively. Comment #3. After all these modifications, the paper will be considered. Response: Thank you very much for your valuable comments and they certainly improved our paper for publication. Attachments Attachment Submitted filename: Response to reviewers 2.docx https://doi.org/10.1371/journal.pone.0270970.r004
22 Jun 2022 Decision Letter - Chakrabhavi Dhananjaya Mohan, Editor Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines. PONE-D-21-32928R2 Dear Dr. Nor Rashid, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Chakrabhavi Dhananjaya Mohan, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0270970.r005
Formally Accepted
29 Jun 2022 Acceptance Letter - Chakrabhavi Dhananjaya Mohan, Editor PONE-D-21-32928R2 Morindone from Morinda citrifolia as a potential antiproliferative agent against colorectal cancer cell lines Dear Dr. Nor Rashid: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Chakrabhavi Dhananjaya Mohan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0270970.r006

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